The development of High Throughput Screening (HTS) assay procedures for the identification of potential drug candidates has established the need for large numbers of cells for each type of test. As a consequence there is a significant challenge to overcome the “culturing burden” to enable multiple billions of cells to be grown for each screen. Anchorage-dependent or adherent cells are of particular interest to the drug industry as many different cells, including most cells derived from solid tissues, require a surface to grow and differentiate. The use of support particles or microcarrier beads (e.g. CYTODEX™ particles (GE Healthcare)) in cell culture methods has improved the yields of adherent cells by increasing the surface area for growth. A range of microcarrier beads are available including dextran (e.g. CYTODEX™) and polystyrene beads coated with a variety of polymers (e.g. gelatine, collagen, FACT, ProNectin F; Sigma-Aldrich).
Microcarrier beads for cell culture provide a large surface area for growing adherent cells to very high cell densities. Maximum cell densities are dependant on the efficient seeding of cells onto the microcarriers. Typically at least 80% of beads should be coated with 5 to 15 cells per bead at the start of the culture phase. To attain this criterion requires a controlled cell seeding phase in which cells and beads are in an environment having both static and dynamic phases. The cells and microcarrier beads settle at different rates: the beads being denser than cells settle before them. The microcarriers will settle to form a bed at the bottom of the culture vessel, the depth of the bed being dependent upon the number of microcarrier beads present. As cells begin to settle, the beads located near the top of the bed will be most likely to receive cells. The cells will begin to attach within minutes, typically within 5 to 60 minutes. However, if the environment were to remain static, non-uniform coating of microcarriers would occur, the microcarriers at the top of the bed being coated with many more cells than beads located within or at the bottom of the bed volume which may receive very few or even no cells. It is therefore necessary to turn over or agitate the microcarrier bed in order to expose the surface area of all the beads to the available cells. Since adherent cells are generally only loosely attached to microcarrier beads at the early stage of seeding, great care must be taken to minimise excessive shear forces which could result in cell detachment and/or cell injury. This can be a problem if a significant bed volume of microcarrier beads has to be turned over.
The problem of maintaining microcarrier beads in liquid suspension whilst minimising shear forces has been addressed previously. For example, U.S. Pat. No. 4,382,685 (Pearson) describes an apparatus for stirring particles in a liquid medium. The stirrer is a rod with a bulbous tip which describes an orbital path in the vessel to sweep out an annular trough or channel formed in the bottom of the vessel. The apparatus is designed for stirring microcarriers in a liquid culture medium to maintain them in uniform suspension at low stirring speeds to avoid damage to growing cells. U.S. Pat. No. 4,355,906 (Ono) describes an apparatus which includes a rotatable stirrer for maintaining microcarrier beads in suspension in cell culture. The stirrer assembly includes at least one blade radially disposed with respect to the vessel axis and is connected to the assembly adjacent a magnet for rotation about the axis. U.S. Pat. No. 4,512,666 (O'Connell) relates to suspended magnetic stirrers, in particular, a suspended magnetic impeller the height of which can be adjusted by means of a movable bearing. See also U.S. Pat. No. 5,267,791 (Christian et al); U.S. Pat. No. 4,634,675 (Freedman et al).
EP 254494 B1 (Davidson et al.) provides a description of known impellers in use for stirring liquids. These include impellers known as the “disk turbine” or “Rushton” impeller in which each paddle of the impeller generally consists of a rectangular-shaped sheet which is mounted around the periphery of a disc or spindle. Other forms of known impeller include the standard “marine” impeller having up to three or four curved blades fixed around a central spindle; and the pitched blade impeller having strip-like blades extending radially outwards from a spindle, each of which is mounted at an angle to the axis of the of the shaft. In operation, liquid is propelled away from the impeller in parallel to the axis of the shaft thereby generating circulation loops within the vessel. Furthermore, standard impellers, such as marine, Rushton or pitched blade are incapable of lifting beads into suspension without the use of high rotational speeds, typically in excess of 60 rpm (revolutions per minute). As a consequence of the high rotational speed and extended time required to lift a large bed volume of microcarriers beads, cells can be damaged and loss of adhesion between cells and microcarrier beads can occur due to excessive shear forces.
JP61074564 (Toyo Boseki) describes an impeller which can be used for the uniform agitation of cells even at low rotational speed and to facilitate the separation of cells from microcarriers after the completion of culture growth. The impeller comprises alternating flat and curved blades, the flat blade forming a radial flow perpendicular to the rotary shaft, and the curved blade forming an axial flow parallel to the rotary shaft.
US 2004/0174769 (Weetman) discloses impellers which are adapted for use in surface aeration of liquids in a tank when disposed on the surface of the liquid in the tank. The aeration efficiency of the impeller is improved by curving the top portions of its and providing an opening or a slot on the blades through which a portion of the liquid may pass.
U.S. Pat. No. 5,277,550 (Kato et al.) relates to agitating vanes for use in a device for agitating and mixing liquid or gases in food processing and the chemical industries. The vanes are designed to produce efficient agitation without damaging microorganisms by reducing eddy flows and peeled-off eddy flows. This effect is achieved by mounting several curved or auxiliary vanes at right angles to a flat vane in a spaced-apart relationship. While such vanes or impellers may be employed for agitating microorganisms, they would not be suitable for use with more sensitive cells such as mammalian cells.
U.S. Pat. No. 5,316,443 (Smith) discloses liquid mixing impellers particularly designed for the chemical processing industry which provide a generally axial flow when rotated in a first direction and a generally radial flow when rotated in the opposite direction. The blades have at their leading edges a curved and folded back section of relatively short chordwise extent, so as to form a concave pocket immediately behind the leading edge.
U.S. Pat. No. 5,297,938 (Von Essen et al.) relates to hydrofoil impellers for use in mixing apparatus which have a high efficiency liquid pumping action and enhanced power stability. The hydrofoil impellers disclosed in the document would not be suitable for use in a bioreactor or cell separator because this pumping action would injure the cells.
SU1114696 (Karpovich et al.) discloses a microorganism cultivating apparatus for hydrogen acid forming bacteria. Two flat bladed mixers agitate the gas-liquid mixture and separate, discrete vertical plates having curved ends are used to disperse the mixture and organise its movement through the circulation contour.
Thus, although the importance of achieving suitable agitation in an environment of low shear force has been recognised, it has been found that none of the impellers described in the prior art provide a solution to this problem. Indeed, none of the prior art documents described above offer a means to completely overcome the requirement to efficiently lift cells and microcarrier beads in a manner that turns over the entire population of cells and/or beads in an environment of low shear force, thereby minimising cellular injury and ensuring that low affinity interactions between cells and beads is retained. The present invention addresses this need by the provision of an impeller device that efficiently and rapidly lifts a bed of settled cells and microcarrier beads at low rotational speeds. Cell growth and concomitant yield is therefore improved by maintaining the culture in an environment of low shear force.
Another problem encountered in the large scale production of adherent cells which are grown on microcarrier beads is that of separating the cells from the beads at the end of the process. The cells are often treated with enzyme solutions, such as trypsin, at the end of the process to facilitate separation from the beads. However, enzymic means are seldom sufficient on their own to effect this separation, and some form of physical agitation is also required to increase yields of the cells. Care must be taken in applying this agitation otherwise the cells will be injured in the process. In one aspect, the present invention addresses this problem by the use of an impeller which can agitate cells at low rotational speeds.